Dear Kathryn & Wolfgang

I have done tissue culture since 1972, and have previously established departmental media mass-production facilities. I understand bicarbonate buffer chemistry quite well, as well as all the other ways in which media is buffered. Moreover, my first hand experience over many years has taught me more than any theory can explain. However I'll try to elaborate further.

The earth's atmospheric CO2 concentration is only 0.04%. In contrast, CO2 incubators are generally set at 5-10% depending on the buffer system. The average humidity over land is quite low, whilst humidity inside the incubator must always be 100% saturation. Opening incubator doors too often thus allows humidity to escape, and consequently vented culture vessels will dry out until humidity is reestablished. This means the salt concentration of the media in which your cells are growing increases, which can kill cells. Even leaving the outer insulating doors open for too long allows the inner glass doors to act as a condenser, and the humidity will drop even though the temperture will remain constant. Because of the relatively high CO2 concentration inside the incubator, this humidity is more like acid rain (carbonic acid). In this regard humidity acts like a CO2 sink to maintain stable CO2 levels, so loss of humidity also means you waste CO2.

CO2 is more soluble in cold water, which is why highly carbonated drinks go flat when warm and open to the air. CO2 gas dissolves in water to produce carbonic acid, and sets up a bicarbonate-carbonate equilibrium that determines dissolved CO2 concentration at a given temperature. Cold drives the reaction to the right, warmth drives it to the left.

CO2 + H2O ↔ H2CO3 ↔ H+1 + HCO3-1 ↔ 2H+1 + CO3-2

carbonic acid bicarbonate carbonate

Carbonic acid is a weak acid and doesn't dissociate its protons easily. Most dissolved CO2 remains as undissociated carbonic acid, or exchanges with atmospheric CO2. Despite warmth, the incubator's CO2 concentration drives the reaction to the right. Culture media formulations are optimised for given CO2 settings (usually 5%) and temperatures (usually 37C). However the humidity must always be 100%, because water is an essential part of the above reaction. I used to grow tumor cells in tightly capped bottles, in a warm room (no CO2 incubator required). After passaging cells, the media would turn alkaline. Over the next few days lactic acid and CO2 produced by the rapidly growing cells would bring the pH back down, at which point the monolayer was confluent and the cells were ready for passage again.

In sodium bicarbonate-buffered media, the bicarbonate concentration is in vast excess of what would occur from dissolved atmospheric CO2. As long as the system is closed (sealed bottle) and CO2 cannot escape, the original pH is maintained. Inside the CO2 incubator, the pH equilibrium is maintained because that's the way it's been formulated. However when open to the air, excess bicarbonate drives this equilibrium reaction to the left and CO2 gas escapes (media goes flat). The result is is a loss of protons (acidity), so the media goes alkaline. Phenol red in DMEM is a sensitive indicator in the pH 6-9 range (orange-purple).

Because tissue culture media contain sodium bicarbonate, there is a new equilibrium reaction established which modifies (buffers) the above reaction.

2 NaHCO3 → 2 Na+1 + 2 HCO3-1 ↔ 2 Na+1 + CO3-2 + CO2 + H2O

The presence of both sodium and bicarbonate ions means that when CO2 escapes, the reaction is driven to the right and sodium carbonate is produced. When CO2 dissolves in media, the reaction is driven to the left. Once the correct amount of bicarbonate has been added, the pH of flat alkaline media should only be adjusted by bubbling CO2 gas through it, not by addition of HCl.

While it is true that bicarbonate ions may be lost, as they are composed of just CO2 and H2O, all sodium ions remain in solution. The buffering ability of sodium bicarbonate allows for dissolved CO2 to be gained (carbonic acid) or lost (sodium carbonate). Carbonate solutions are more basic than bicarbonate on a mole for mole basis. Carbonate ions can neutralize two protons, whilst bicarbonate can only neutralize one. Besides, it's really not this simple, as there are other buffers including serum which stabilize the pH.

To understand bicarbonate buffers is to understand global warming. The earth's oceans are full of insoluble calcium carbonate - think sea shells and coral reefs. As increased atmospheric CO2 drives formation of acidic oceans, this carbonate sink will be dissolved as it becomes bicarbonate. Whales migrate to cold waters to feed because there is more dissolved CO2 to support photosynthesis by phytoplankton, the base of the food chain. And so on, and so on.

I calibrate my pH meter..Moreover the colour of the media changes from blood red to slightly purple. I am using DMEM powder media..

I checked my media for contamination and also prepared media thrice.I am using sodium bicarbonate composition of 1.5 gm/lt at 5% co2.

Hi Deeksha

You need to add HEPES buffer in addition to sodium bicarbonate to maintain the PH. For DMEM you can use the following method of preparation.

1. In a 1000-ml sterile beaker, a packet of the DMEM is dissolved in 600-ml of sterilized distilled water.

2. Then, a 5.9575 gm of HEPES buffer is added to the media, stir on a magnetic stirrer.

3. Add 3.7 gm of sodium bicarbonate, 10-ml of antibiotic, 10-ml of essential amino acid, and 10-ml of non-essential amino acid.

4. Stir the components for 10 minutes.

5. Adjust the pH of the media to 7.2 by using diluted solutions of NaoH (1N) or Hcl (1N) and the pH meter.

6. Finally, complete the volume of the media to 1000-ml by sterilized distilled water using a graduated cylinder.

7. Sterilize the media by using 0.22 µm Milli-pore filter , which traps most of the microbial contamination found in the prepared media.

8. Keep in 4 C.

9. Add serum on aliquots before hand

Good Luck

Note added (perhaps not necessary due to Gabers hint (:-)) ):

i) http://www.researchgate.net/post/Adding_HEPES_to_medium

find: Re by: Alan Fairlamb · University of Dundee

Sodium bicarbonate is a buffering agent, provided there is an atmosphere of 3-5% carbon dioxide. If not, bicarbonate will pick up hydrogen ions forming carbonic acid which dissociates releasing carbon dioxide into the gas mixture. The net result is that the culture medium becomes more alkaline. This is also observed when filtering media under negative pressure. Having 20 mM HEPES, or another Good buffer such as TES, present ameliorates this effect. Sigma sells many versions of DMEM, some of which contain HEPES, others that do not, so it is possible that authors have not been specific in describing their method.

Jun 18, 2012 & ff,

[ reference for Eagle, H. Science 174: 500-3 (1971) =

http://www.ncbi.nlm.nih.gov/pubmed/5110427

ii) cf.: http://www.sigmaaldrich.com/life-science/cell-culture/classical-media-salts/dmem.html, look for:

D 1152: Dulbecco's Modified Eagle's Medium HEPES Modification /

Powder

With L-glutamine and 4500 mg/L glucose and 25 mM HEPES

Without sodium bicarbonate, supplement with 3.7 gm/L of sodium bicarbonate

Formulated at 17.4 grams of powder per liter of medium

Cell culture tested

Same as D 5648, except that it is supplemented with HEPES. HEPES supplementation increases the buffering capacity of the medium. Recommended for cells that require a higher pH (7.3-7.6) for optimal growth."

perhaps intersting with this also another article found at: http://www.sciencedirect.com/science/article/pii/S0014482784712146

(pH Changes in Pulsed CO2 Incubators Cause Periodic Changes in Cell Morphology.1994) and perhaps also

iii) http://onlinelibrary.wiley.com/doi/10.1002/pmic.200300395/full (2003) pointing to perhaps "subclinical" changes in behaviour of cultured cells varying pH of culture medium.

Best regards, Wolfgang

Thanks a lot Dr. Gaber and Dr. Muss

Dear Deeksha, you're welcome...Best of luck, regards, Wolfgang

Dear Deeksh

Thanks very much

Dear Dr. Muss and Dr. Ramadam,

It is evident that you have expertise in this area, and I thought that rather than starting a completely new thread I would ask my question here, as it is very well related to what has been said above.

The question is this:

For bicarbonate/CO2 buffered (HEPES free) DMEM media being stored in the fridge, the immediate CO2 environment is just atmospheric CO2 concentrations (i.e. low). Because of this, the bicarbonate should pick up H+, form carbonic acid, dissociate into H2O + CO2 and release CO2 into the air in the bottle.

This results in the media becoming more alkaline over time, but would it not also result in a reduction in the moles of bicarbonate present in the media? So over time, the concentration of bicarbonate in the media would decrease?

Would this then have the effect of reducing the overall buffering capacity of the media over time?

Based on this, if you then used aged media in cell culture at 5% CO2, would aged media acidify more under these elevated CO2 conditions than media made up fresh, having lost some of it's buffering capacity? (all else held constant)

I have done the same experiment several times and it seems like the pH of the media (even when in the incubator) was changing over time. It would make more sense with my results if it was becoming more acidic. Could the mechanism described above explain this phenomenon?

Many many many thanks if you have an answer for me - I need it for my honours thesis!

Kind Regards,

Kathryn Parker

Apologies to Dr. Ramadan for the typo above

Good morning,

Dear Kathryn, thank you for your acknowledged info above I unfortunately could see / find only today (was out of Lab for the last week).

Your suggestions are really interesting, but I have to confess that - as an electron microscopist - dealing with buffer solutions maintaining cellular milieu at a reasonable "physiologic" balance during the "chemical fixation" I am not able to give you reliable answers here and now. If I had time now I would think about hard and perhaps I could come to a solution. But unfortunately - and since I am NOT a chemist (or biochemist) - the chance for answering correctly nevertheless would be minimal.

On the other hand - especially with Tyrode's solutions as well as with my phosphate buffers (especially the stock solutions) performed sometimes (when I got aware of it and measured with the pH-meter!) poor with respect to their "holding" their pH-value (after some day/s) which initially was adjusted correctly .

Reading again right now your initial question: you are measuring your medium when ready (at room temperature -23-24 degr.C - I guess- and shows pH 7.2). Then you store the medium in the fridge over night and let warm up to RT (and did you measure the pH at that point??), followed by raising the temperature to 37 degr.C, resulting in a final measure of pH 7.8.

I am not quite sure (at least for the bicarbonate buffer combination you use) whether the raising of temperature from RT - say - 23/24 degr.C to 37 degr.C (= over 10 degr.C) would result in the higher pH value of 7.8?? (for "plain" PO4 buffer I have or could find values....)....for now, happy thinking and with my best wishes and regards, Wolfgang

It is normal for culture media to turn alkaline over time upon storage, especially as the bottle is opened and closed. DMEM high glucose usually has extra bicarbonate buffer to counteract the lactic acid that will be produced by rapidly growing cells, so it is particularly true of this media. Bicarbonate is not the only buffer - there are phosphates and amino acids that all interact together. Once alkaline media equilibrates in cultures in a CO2 incubator, the pH will be fine. However I do not use media which has gone purple in the bottle, as I believe certain components may irreversibly denature.

Something important to keep in mind: CO2 is more soluble in cold water than warm water. Pre-heating media in water baths (which is quite common in labs) not only drives off CO2 (making the media more alkaline), but also destroys glutamine. Moreover, water baths are a notorious source of microbial contamination. When passaging cells, it is not necessary to pre-heat media. Just stand at room temperature long enough to take the chill off. An exception is when changing media on established monolayers, or experiments that require continuous growth. Adding cold media will shock the cells, and it will be a while before they recover. In this case, pre-warm only as much media as needed.

Good day...

@Kathryn...I know that YOUR complex question was not answered reliably. On the other hand I added a paragraph in my reply to you, namely:

"Reading again right now your initial question:" so I apologize that this was addressed @Deeksha Pal, the initial requester...

Again @ Kathryn: I will think about your comment again..for now I guess that one should search for parameters in of especially the cited bicarbonate buffer example.

You said:

....... perhaps logic thoughts, but the chemistry & reaction mechanisms, especially when "aging" for a complex buffering system like the DMEM+Hepes (cf. Gaber Ramadans post) are hazy figures (at least for me). The question then of "for how long such a - permanently - "aging" buffer can maintain its buffer capacity.... because, IMHO, if the buffer doesnot / is not able to maintain the intrinsic pH of the solution then it cannot be called "buffer" any more.

I really hoped anybody with wisdom and real knowledge about buffer systems would chime in as early as possible.

One specific question I have: you said:

.... guessing you meant the buffer medium will raise in alkalinity?? (or am I misinterpreting your information?)

If I can find some info I will come back here to let you know....

Best wishes and regards, Wolfgang

Wow thank you both for these extensive answers!

In regards to the temperature at which the pH was measures/adjusted. Indeed, it was room temperature, about 22 degrees I would say. The the temperature dependence of pH does make things tricky when trying to measure what the pH really was in the incubator!

In regards to the pre-heating of media. It is very interesting that heating the media may have accelerated the loss of CO2 and therefore HCO3-. It so happens that I am using a monolayer, so as you say I do pre-heat the media in a water bath. In future I will pay close attention to the temperature the media is at to try and minimise this effect.

Wolfgang, in response to your last post I agree that this whole 'aging' process is probably more complicated than what I am accounting for with my 'the-bicarbonate-concentration-is-decreasing-hypothesis'.

To further clarify my question I want to work out whether aged media might become *more acidic* than fresh media when placed in a 5% CO2 incubator.

pH is in the incubator is related to HCO3- concentration by a modified version of the Henderson-Hasselbalch equation:

pH = 6.1 + log( [HCO3-]/(kH*PCO2) )

where kH is Henry's constant for CO2 dissolving into liquid (at 37degreesC) and PCO2 is the partial pressure of CO2 in the incubator.

My thought is that if one uses 'aged' media in the incubator in which the HCO3- concentration has decreased over time, the pH would be lower than for media made up fresh.

So the question becomes: does the HCO3- concentration has decrease over time,

and

if not, what other possible explanations might account for media becoming more acidic in sequential experiments.

I really appreciate people doing all this thinking on my behalf!

Kind regards,

Kathryn

Dear Kathryn & Wolfgang

I have done tissue culture since 1972, and have previously established departmental media mass-production facilities. I understand bicarbonate buffer chemistry quite well, as well as all the other ways in which media is buffered. Moreover, my first hand experience over many years has taught me more than any theory can explain. However I'll try to elaborate further.

The earth's atmospheric CO2 concentration is only 0.04%. In contrast, CO2 incubators are generally set at 5-10% depending on the buffer system. The average humidity over land is quite low, whilst humidity inside the incubator must always be 100% saturation. Opening incubator doors too often thus allows humidity to escape, and consequently vented culture vessels will dry out until humidity is reestablished. This means the salt concentration of the media in which your cells are growing increases, which can kill cells. Even leaving the outer insulating doors open for too long allows the inner glass doors to act as a condenser, and the humidity will drop even though the temperture will remain constant. Because of the relatively high CO2 concentration inside the incubator, this humidity is more like acid rain (carbonic acid). In this regard humidity acts like a CO2 sink to maintain stable CO2 levels, so loss of humidity also means you waste CO2.

CO2 is more soluble in cold water, which is why highly carbonated drinks go flat when warm and open to the air. CO2 gas dissolves in water to produce carbonic acid, and sets up a bicarbonate-carbonate equilibrium that determines dissolved CO2 concentration at a given temperature. Cold drives the reaction to the right, warmth drives it to the left.

CO2 + H2O ↔ H2CO3 ↔ H+1 + HCO3-1 ↔ 2H+1 + CO3-2

carbonic acid bicarbonate carbonate

Carbonic acid is a weak acid and doesn't dissociate its protons easily. Most dissolved CO2 remains as undissociated carbonic acid, or exchanges with atmospheric CO2. Despite warmth, the incubator's CO2 concentration drives the reaction to the right. Culture media formulations are optimised for given CO2 settings (usually 5%) and temperatures (usually 37C). However the humidity must always be 100%, because water is an essential part of the above reaction. I used to grow tumor cells in tightly capped bottles, in a warm room (no CO2 incubator required). After passaging cells, the media would turn alkaline. Over the next few days lactic acid and CO2 produced by the rapidly growing cells would bring the pH back down, at which point the monolayer was confluent and the cells were ready for passage again.

In sodium bicarbonate-buffered media, the bicarbonate concentration is in vast excess of what would occur from dissolved atmospheric CO2. As long as the system is closed (sealed bottle) and CO2 cannot escape, the original pH is maintained. Inside the CO2 incubator, the pH equilibrium is maintained because that's the way it's been formulated. However when open to the air, excess bicarbonate drives this equilibrium reaction to the left and CO2 gas escapes (media goes flat). The result is is a loss of protons (acidity), so the media goes alkaline. Phenol red in DMEM is a sensitive indicator in the pH 6-9 range (orange-purple).

Because tissue culture media contain sodium bicarbonate, there is a new equilibrium reaction established which modifies (buffers) the above reaction.

2 NaHCO3 → 2 Na+1 + 2 HCO3-1 ↔ 2 Na+1 + CO3-2 + CO2 + H2O

The presence of both sodium and bicarbonate ions means that when CO2 escapes, the reaction is driven to the right and sodium carbonate is produced. When CO2 dissolves in media, the reaction is driven to the left. Once the correct amount of bicarbonate has been added, the pH of flat alkaline media should only be adjusted by bubbling CO2 gas through it, not by addition of HCl.

While it is true that bicarbonate ions may be lost, as they are composed of just CO2 and H2O, all sodium ions remain in solution. The buffering ability of sodium bicarbonate allows for dissolved CO2 to be gained (carbonic acid) or lost (sodium carbonate). Carbonate solutions are more basic than bicarbonate on a mole for mole basis. Carbonate ions can neutralize two protons, whilst bicarbonate can only neutralize one. Besides, it's really not this simple, as there are other buffers including serum which stabilize the pH.

To understand bicarbonate buffers is to understand global warming. The earth's oceans are full of insoluble calcium carbonate - think sea shells and coral reefs. As increased atmospheric CO2 drives formation of acidic oceans, this carbonate sink will be dissolved as it becomes bicarbonate. Whales migrate to cold waters to feed because there is more dissolved CO2 to support photosynthesis by phytoplankton, the base of the food chain. And so on, and so on.

Dear Gerald,

I thank you so much for the thorougly compiled reply & really understandable explanation(s) on the mechanism. I admit that I have learnt much with regard to bicarbonate buffer system in cell culture. And, for sure, such expert opinions as yours presented at /on fora/forums like ResearchGate are the quintessence on/of the success of this specific platform.

Thanks again, best regards and wishes,

Wolfgang

You're most welcome, Wolfgang. You were very helpful to Kathryn and she was really appreciative, so I felt inspired to make a contribution. Besides, Kathryn is at an Australian university so I have a certain obligation. :-) Hopefully others will learn from this as well.

Regards

Gerry

Dear Gerald, (dear Kathryn),

only short here: that's why I joined ResearchGate and as retiring next year I also feel that we (the elder ones) do have some kind of obligation, especially to the "young(er) scientists" to help and stimulate their struggle(s) and willingness to become well educated and thinking researchers in the future (and, naturally, independent of their RG-score!). And last but not least: I second 100% your last sentence! Thank you again for your valuable input here, best regards, Wolfgang

Dear Gerald and Wolfgang,

Thank you Gerald for your great answer! It has certainly cleared up a few things: I will be sure to keep the caps on my media bottles tight, this was something I probably wasn't doing enough at some points.

So thanks to both of you, your answers have been very helpful. I am new to this forum but so far all my experiences with it have been very good, I think it is a big success!

Kind regards and many thanks again,

Kathryn

In sodium bicarbonate-buffered media, the bicarbonate concentration is in vast excess of what would occur from dissolved atmospheric CO2. As long as the system is closed (sealed bottle) and CO2 cannot escape, the original pH is maintained. Inside the CO2 incubator, the pH equilibrium is maintained because that's the way it's be

Kunal Pal,

Below is what Gerald Waneck wrote:

"In sodium bicarbonate-buffered media, the bicarbonate concentration is in vast excess of what would occur from dissolved atmospheric CO2. As long as the system is closed (sealed bottle) and CO2 cannot escape, the original pH is maintained. Inside the CO2 incubator, the pH equilibrium is maintained because that's the way it's been formulated."

When comparing that to what you pasted, are you really contributing anything at all? You couldn't even be bothered to finish his sentence.

Dear Ron Hrstka (apologies, the 'Mention' function doesn't work for me),

thank you for pointing to Kunal PAL's plagiate from Gerald WANECKS post/reply # [016] 5 years ago.

As a consequence I withdraw my earlier recommendation for Kunal PAL's reply [022] right now.

I honestly add that normally I don't read former answers in full (when I have followed the thread every reply given...), especially if the interim is more than 4 .5 years....

So thank you again, dear Ron, and my best wishes for your career at the Mayo-Clinic (assuming Rochester, USA). W.H.M.

Wolfgang Muss, oddly the mention function does not work for me either. If Kunal Pal's cut/paste answer had been a one off, I would have ignored it, but he has done it dozens of times. I first noticed it when he copied one of MY answers. I have reported some of his posts and nothing was done, so I took the petty route and began commenting on his comments during my coffee breaks.

Crosswords or sudoku might be more rewarding.